Abstract The explosive spread of Zika virus (ZIKV) infection and its associated fetal microcephaly and other birth defects present an urgent need for highly sensitive and specific diagnostic tests, particularly for pregnant women. ZIKV belongs to the genus Flavivirus, which includes several pathogenic mosquito-borne viruses of different serocomplexes, such as the four serotypes of dengue virus (DENV), West Nile virus (WNV), Japanese encephalitis virus (JEV), yellow fever virus (YFV), and ZIKV. The current CDC guidelines for laboratory tests of ZIKV include a positive RT-PCR test to confirm ZIKV, and a negative IgM test to exclude ZIKV. Since the majority of ZIKV infections are asymptomatic and many seek ZIKV tests beyond the period of detectable RNA, serology remains a critical component of ZIKV diagnosis. Due to the cross-reactivity of antibodies against the envelope (E) protein with other flaviviruses, positive or equivocal IgM tests based on E protein require time-consuming neutralization tests, which can only confirm those acquiring ZIKV for the first time, greatly limiting the usefulness of E protein-based serological tests in flavivirus-endemic regions. Recently, we discovered that antibodies to non-structural protein 1 (NS1) and premembrane (prM) protein recognize NS1 and prM proteins of members within the same serocomplex but not those of different serocomplexes, and that combined two NS1-ELISAs can distinguish primary ZIKV infection, ZIKV with previous DENV infection, and secondary DENV infections. Our long-term goal is to develop serological tests to accurately delineate past and present flavivirus infections. The objective is to develop highly sensitive and specific rapid serodiagnostic tests for ZIKV infection in flavivirus- endemic regions. The central hypothesis is that detection of anti-NS1 and anti-prM antibodies to different flaviviral serocomplexes and their combination can distinguish infections caused by flavivirus of different serocomplexes. The first aim is to develop and validate IgG ELISA based on recombinant NS1 and pr proteins to detect and distinguish ZIKV infection from other mosquito-borne flaviviral infections. The second aim is to develop and validate IgM ELISA based on recombinant NS1 and pr proteins. Thirteen comprehensive panels of post-convalescent-phase serum or plasma from 8 confirmed flaviviral infections or vaccination, including infections by four DENV serotypes (primary and secondary infections), WNV, JEV, ZIKV (primary and those with previous DENV infections), and YFV-17D vaccination together with flavivirus-nave samples will be tested. The significance of the proposed research rests on the development of sensitive, specific and convenient IgG and IgM tests to distinguish ZIKV from other mosquito-borne flaviviruses. These tests can be directly applied to routine ZIKV serodiagnosis and serosurveillance study, and be further developed into high-throughput assays or point-of-care rapid tests. The proposed study is innovative as it focuses on combination of two promising viral antigens (NS1 and pr), one of which has never been recognized to be specific to ZIKV, to develop serological tests for ZIKV and combination of multiple assays to distinguish different ZIKV and DENV infections.